BR112014001544B1 - TAPE WITH INDIVIDUAL PILLOW FOR AN IMPROVED SIDE FLOW TEST AND CHROMATOGRAPHIC TEST - Google Patents

Abstract

tape with individual pad for an improved lateral flow test and test device using it. the present invention relates to a strip for an improved lateral flow assay of a biological sample in a single plane and a lateral flow chromatography assay using a test containing the same. the fixture of the present invention consists of an individual cushion, which can improve the lateral flow test by providing an easy and simple procedure and an evident visual reading. the tape of the present invention consists of a sample application zone (sample) and a resultant reagent zone, in which the reaction mixture is deposited (reagent), which are on the same plane. furthermore, the present invention provides a chromatographic method in which hemoglobin is separated from the analyte by means of differential solid-phase chromatography. any interference with hemoglobin detection of the result is removed by the present invention. the present invention provides advantages, including an easy and simple procedure with a quick and evident response.

Description

CROSS REFERENCE FOR RELATED INVENTION PATENT APPLICATIONS

[001] The present application for a patent claims priority over the US provisional application for a patent with serial number 61/510 762, filed on July 22, 2011, the content of which is considered to be incorporated by reference.

BACKGROUND OF THE INVENTION

[002] Tests performed at the point of care (POC) have become common and very useful diagnostic tools not only in medical care facilities, including hospitals and doctors' offices, but also in other clinical locations, including workplaces or more remote locations. Due to the urgency and purpose of the tests, a simple and effective test device, as well as a method to acquire the results in an economical and time-efficient way, is highly desirable. More specifically, testing in facilities other than clinical care, such as testing in remote clinical sites of rapidly developing epidemic diseases, testing for biological weapon agents in war scenarios, environmental testing for pollutants, or testing on the spot regarding the use of drugs, it should be done in a simple and easy way. Sometimes, as these tests are often performed by individuals with little, if any, experience in clinical diagnostics, these tests at “point of care” sites should be simple, quick and easy to use. In addition, devices are needed in environments with little control under storage conditions, such as in terms of temperature or humidity. Thus, ideally, the device for on-site testing of the point of care requires a minimum amount of equipment and a test method embedded in the device stable under severe environmental conditions, including temperature and humidity, would be desirable.

[003] With the increasing need for testing at point of care sites for an increased number of purposes and environmental variants, there is still a need in the art to provide a method and device for carrying out tests for a biological specimen of an economical and time-efficient way.

SHORT DESCRIPTION OF THE INVENTION

[004] According to a variant, the present invention provides a tape based on an individual pad for a lateral flow test in which the sample application zone (sample) and the resulting reagent zone where the reaction mixture is deposited (reagent) are all on the same plane as an individual pad.

[005] According to another variant, the present invention provides a tape for an enzymatic chromatographic lateral flow assay for a pre-selected analyte, which consists of: an individual pad and a substrate deposited as anhydrous form.

[006] According to yet another variant, the present invention provides an enzymatic chromatographic lateral flow test for a pre-selected analyte, which comprises; (a) providing a test device comprising a tape consisting of: (i) an individual pad and (ii) a substrate deposited as anhydrous form; (b) applying a sample to the individual pad on which the substrate is deposited; (c) allowing the sample to flow through the digestive path to reach the substrate; (d) allowing the sample to react with the substrate to produce a response and (e) identifying and interpreting the response to indicate the presence or concentration of the analyte in the sample.

[007] According to another variant, the present invention provides an enzymatic chromatographic side-flow assay to detect the presence of glucose-6-phosphate dehydrogenase (G6PD), which comprises: (a) providing a test device comprising a individual pad tape consisting of: (i) an individual pad of digestive tract pre-treated with a lysis buffer; (ii) a test mixture comprising glucose-6-phosphate, nicotinamide-adenine-dinucleotide (NADP) or nicotinamide-adenine-dinucleotide (NAD), hydride or electron transfer agent, and a mixture thereof ( iii) a tetrazolium salt, (b) apply the sample to the individual pad of the digestive tract; (c) allowing the sample to flow through the digestive path until it comes in contact with the test mixture; (d) allowing the sample to react with the test mixture to produce a response and (e) identifying and interpreting the response to indicate the presence or concentration of the analyte in the sample.

[008] According to another variant, the present invention is based on a dry format test in which the sample applied in the sample application zone travels to the reaction zone by capillary force, the application zone and the reaction zone being on the same plane. The reagent contains a combination of components including the enzyme and a composition to promote the color of the reaction, which is deposited as an anhydrous form. In the present invention, the sample travels to the reagent zone and is mixed with the reagent. As the sample is mixed with the reagent, the resulting composition will migrate slowly and remain in the resulting reagent zone, while the other components in the sample, such as hemoglobin, move faster than the resulting composition will continue. moving to separate from the desired resulting composition through that capillary force. After reacting with a sample containing the analyte, the resulting reagent zone will display the result as an indicator, such as a color. Retaining the resulting, continuous capillary movement of the other components in the sample will remove any interference from parts of the other components, such as hemoglobin, and improve the identification of the result. The sample moves by capillary force in a liquid contained in the sample, such as blood, or in a buffer solution, which is applied further with the sample.

[009] According to a variant, the present invention provides a rapid diagnostic test (in English RDT) in an anhydrous test on an individual pad tape for a test in a "point of care" location. Side chromatography on an individual pad provides a stable test, a simple process and a quick test result.

[010] According to another variant, the present invention provides a chromatographic method in which hemoglobin is separated from the analyte using differential solid-phase chromatography. The color of the hemoglobin often interferes with the visual reading of the color of the reaction of the substance to be analyzed in red blood cells. The present invention provides a lateral chromatographic method in which the differential migration of samples occurs due to differences in size, viscosity, precipitation of the analyte with reagent, the difference in charge through the porous matrix impregnated with reagents that will interact with the analyte.

[011] The advantages of the invention include the ability to obtain an easy and simple procedure and a clean visual reading free from hemoglobin interference.

[012] Throughout the present patent application, the following terms have the meanings presented below.

[013] The term "biological material", "biological sample" or "sample" means a fluid or tissue extracted from vertebrates, such as whole blood, serum, plasma, saliva, urine and spinal cord fluid.

[014] The term “analyte” means a particular component in a biological material, biological sample or sample that contains a specific activity, which is the purpose of the test to identify one of the results, such as the presence, absence or concentration.

[015] Unless otherwise indicated, all technical or scientific terms used herein have the same meaning as is commonly known to those skilled in the art, to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references are hereby incorporated in their entirety by reference. In case of conflict, this specification, including definitions, should be the reference. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

[016] Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

SHORT DESCRIPTION OF THE FIGURES

[017] Fig. 1 provides a structure of the individual pad tape of the present invention, compared to the conventional two-pad tape.

[018] Fig. 2 provides results of visual comparison tests between the individual pad tape of the present invention compared to the conventional two-pad tape for the G6PD deficiency test. The intensity of visual color identification is measured by the classification provided in the figure.

[019] Fig. 3 provides results of the signal strength comparison test between the individual pad tape of the present invention compared to the conventional two-pad tape using an enzymatic assay for the G6PD test. The intensity of visual color identification is measured by the classification provided in the figure.

[020] Fig. 4 provides visual comparison test results between the individual pad tape of the present invention compared to the conventional two-pad tape for a lactate dehydrogenase test.

[021] Fig. 5 provides signal strength comparison test results between the individual pad tape of the present invention compared to the conventional two-pad tape using an enzyme assay for the lactate dehydrogenase test. The intensity of visual color identification was measured using an aQzen reader.

[022] Fig. 6 provides visual comparison test results between the individual pad tape of the present invention compared to the conventional two-pad tape for an alcohol test.

[023] Fig. 7 provides test results for comparing signal strength between the individual pad tape of the present invention compared to the conventional two-pad tape using an enzyme assay for an alcohol dehydrogenase test. The intensity of visual color identification was measured using an aQzen reader.

[024] Fig. 8 provides signal strength comparison test results between the individual pad tape of the present invention compared to the conventional two-pad tape using an enzyme assay for a lactate dehydrogenase test. The intensity of visual color identification is measured by the classification provided in the figure.

DETAILED DESCRIPTION OF THE INVENTION

[025] The present invention provides a tape for an enzyme-driven chromatographic side flow assay for a pre-selected analyte, which consists of: an individual pad and a substrate deposited as anhydrous form.

[026] According to a variant, the individual pillow of the present invention constitutes a digestive pathway pretreated with a lysis buffer, such as magnesium chloride.

[027] According to another variant, the analyte is selected from proteins, carbohydrates, lipids, nucleic acids in human and animal fluids.

[028] According to another variant, the substrate deposited as anhydrous form is a test mixture that contains a reactive substrate for the analyte, such as glucose-6-phosphate for glucose phosphate dehydrogenase (G6PD), which can still comprise nicotinamide-adenine-dinucleotide phosphate (NADP), hydride transfer agent and a mixture thereof.

[029] According to a particular variant, the analyte is selected from glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase or alcohol.

[030] According to a preferred variant, the analyte is glucose-6-phosphate dehydrogenase (G6PD).

[031] According to another variant, the substrate contains an indicator, such as a coloring compound. It is possible to use, without limitation, many compounds, which can be changed electronically or structurally by contact of the substrate with the analyte and which express the change in any form that can be detected visually or through the use of a measurement method or instrument . Examples of such compounds include, but are not limited to, dyes. Compounds that express different colors often change color due to any chemical change or the state of electrons in the structure. Formazan dyes are chromogenic compounds produced from a reduction of tetrazolium salts by dehydrogenases and reductases. Formazan dyes have a variety of colors ranging from dark blue to deep red to orange, depending on the original tetrazolium salt used as a substrate for the reaction. A listing of tetrazolic salts comprises, but is not limited to, TTC (tetrazolium, tetrazolium chloride or 2,3,5-triphenyl-2H-tetrazolium chloride), INT (2- (4-iodophenyl chloride) -3- (4-nitrophenyl) -5- phenyl-2H-tetrazolium), MMT (3- (4,5-dimethyl-2-thiazolyl) bromide -2,5-diphenyl-2H-tetrazolium), XTT (2 , 3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide), NBT (Nitroblue tetrazolium), MTS (3- (4,5-dimethylthiazol-2-yl) - 5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) or DCPIP (2,6-dichlorophenolindophenol). Some of the tetrazolium salts are more soluble in water than others. By reduction either with enzymes or chemical conditions, such as by reaction with nicotinamide-adenine-dinucleotide hydride (NADH) or with nicotinamide-adenine-dinucleotide phosphate (NADPH) or with any hydride transfer agent, the tetrazolium salt it may change color and may even form an insoluble precipitate. The result can be detected by methods such as, but not limited to, visual inspection, colorimeter, UV detector, spectrophotometer, image analysis reader, etc.

[032] According to a variant, the present invention provides an enzymatic side flow chromatographic assay for a pre-selected analyte, which consists of: (a) providing a test device comprising a tape consisting of: ( i) an individual pillow and (ii) a substrate deposited as anhydrous form; (b) applying a sample to the individual pad on which the substrate is deposited; (c) allowing the sample to flow through the digestive path to reach the substrate; (d) allowing the sample to react with the substrate to produce a response and (e) identifying and interpreting the response to indicate the presence or concentration of the analyte in the sample.

[033] According to a preferred variant, the analyte is an enzyme, such as G6PD.

[034] In accordance with another preferred variant, the present invention provides an enzymatic lateral flow chromatographic assay to detect the presence of G6PD, which consists of: (a) providing a test device comprising a tape consisting of: (i ) an individual pad of digestive tract pre-treated with a lysis buffer; (ii) a test mixture comprising glucose-6-phosphate, nicotinamide-adenine-dinucleotide phosphate (NADP) or nicotinamide-adenine-dinucleotide (NAD), hydride transfer agent and a mixture thereof and (iii) a salt tetrazolium, (b) apply a sample to the individual pad of digestive tract; (c) allowing the sample to flow through the digestive path to contact the test mixture; (d) allowing the sample to react with the test mixture to produce a response and (e) identifying and interpreting the response to indicate the presence or concentration of the analyte in the sample.

[035] The present invention provides a lateral flow test in which the sample application zone (sample) and the resulting reagent zone, in which the reaction mixture (reagent) is deposited, are in the same plane, on a pad individual.

[036] Furthermore, the present invention is based on an anhydrous format test, in which the sample applied in the sample application zone travels to the capillary reaction zone, while the application zone and the reaction zone are on the same plane. The reagent contains a combination of components to promote a colored reaction, which is deposited as an anhydrous form. According to the present invention, the sample travels to the reaction zone and mixes with the reagent.

[037] As the sample is mixed with the reagent, the resulting composition will precipitate, moving slowly and remaining in the resulting reagent zone, while the other components in the analyte, such as hemoglobin, will continue to displace up, separating from the desired resulting material by means of capillary force. After reaction with the sample containing the analyte, the resulting reaction zone will present the result with an indicator, such as color, which can be detected visually. At the same time, a reverse reading is also possible: after reaction with a sample containing the analyte, the resulting area of reaction with an indicator, such as color, would disappear. (Sample that contains no analyte: the color remains in the result area). Retaining the resulting, continuous capillary movement of the other components in the sample will remove any interference by the other substances to be analyzed, such as hemoglobin, and improve the identification of the result. The sample travels by capillary force in a liquid contained in the sample, such as blood, or in a buffer solution that can be applied further with the sample.

Individual cushion tape

[038] The present invention provides an individual pad tape device, where the sample application zone and the resulting reagent zone are all on the individual pad. The test carried out on an individual pad will provide a uniform migration of the substances to be analyzed and produce consistent results by eliminating non-uniform migration of the sample through multiple layers. For example, when a blood sample is applied to the individual pad strip which is pretreated with lysis buffer, hemoglobin can be easily separated from the resulting material.

[039] In addition, it is possible to increase the sensitivity in the individual pad tape device by increasing the current amount of analyte that reacts with the reagent instead of the amount of analyte, as the sample will travel across the same surface instead of passing through different media or layers. In addition, using the individual pad tape device, the production cost will be lower and the process will be simpler than in the case of a conventional multilayer test.

[040] For example, the BinaxNOW®G6PD test has a tape that consists of two different pads that are a sample pad and a reaction pad. Samples with normal G6PD activity produce a distinct color change. The red color of the sample is changed to a brown / black color in the upper half of the reaction pad. The color of the reaction product in a blood sample does not eliminate the color of the hemoglobin; therefore, the brown / black color does not show an evident difference from the red color of the blood. Unlike the bi-phase or multi-phase and batch system used in other devices, including the BinaxNOW®G6PD, the present invention uses a single phase continuous chromatography. The test strip consists of an individual pad and continuous chromatography provides easier separation of the resulting product with a dye, such as formazan dye, from the hemoglobin in the strip than in two-stage chromatography.

[041] The present invention consists of three areas, the sample application zone (sample), the reagent zone and the resulting zone. The reaction zone is the area where the combination of components to promote the colored reaction is deposited as an anhydrous form, the resulting zone is a visual reading area and the sample zone is an area over which the sample is applied. Since these zones are made up of the same matrix, the individual pad, a continuous shape is obtained that allows uniform migration of the substance and produces consistent results by eliminating the possibility of non-uniform migration of the sample through multi-layer . In addition, sensitivity can be increased by increasing the current amount of analyte that passes through the multi-layers. Furthermore, the shape of the present invention lowers the cost of the product and makes the product process easier than conventional multi-layer testing.

Lateral flow chromatographic assay

[042] The analyte reacts with the reagent using various reactions, such as, immunological reaction between antibody-antigen reaction, enzyme-substrate reaction, protein-substrate conjugation or formation of substrate-substrate complex.

[043] Although immunological assays often use a side flow chromatographic assay, the side flow chromatographic assay is not commonly used for enzyme assays. A side flow enzyme assay identifies a particular enzyme for its presence or absence and the concentration using a reaction with a substrate and detects the reaction using an indicator, such as a dye, which can express a certain color after reaction with a certain chemical , such as a hydride, produced by the reaction between the enzyme and the substrate.

Glucose-6-phosphate dehydrogenase (G6PD)

[044] A specific variant of the present invention provides an enzyme-based assay, such as the direct detection of the presence of an enzyme in the sample, such as G6PD in a blood sample, using the metabolite activity from the reaction between the enzyme and its substrate, which can reduce the dye to express certain indicators, such as colors. The dye molecule, such as a tetrazolium compound, can be reduced to form an insoluble formazan. The insoluble formazan will have a purple color that can be easily identified visually without using any specific equipment.

[045] The human enzyme glucose-6-phosphate dehydrogenase (G6PD) plays a critical role in human biochemistry. It is part of the oxidative pathway of pentose, in which it acts to minimize the oxidative attacks of free radicals in cells providing reduction equivalents. For example, G6PD converts glucose-6-phosphate to 6-phosphonoglutonate, thereby releasing a proton that reduces nicotinamide-adenine-dinucleotide (NADP) phosphate to NADPH, a reduced form of NADP. NADPH initiates a set of reactions downstream that ultimately reduce the free radical of oxidizing agents and render many of them ineffective in normal human biochemistry.

[046] G6PD is present in most human cells, but it is present in a higher concentration in red blood cells, which, as one of its main functions, act as oxygen transporters, being, therefore, particularly susceptible to oxidative attacks. This enzyme helps to protect red blood cells from oxidative damage and premature destruction. The effectiveness of G6PD is quite high, as reflected by the fact that, during normal activity, less than 1% of its capacity is used to combat and prevent unwanted oxidative reactions. However, when strong oxidizing agents, such as members of the quinine class of anti-malarial drugs have to be introduced into the human body, the need for rapid production of reducing agent is greatly increased.

[047] Several mutations of the gene encoding G6PD are known, which decreases the effectiveness of enzymes in the biochemistry of individuals who process such a mutation in both halves of their genome, causing the amount of their G6PD to remain at the same level as in people with the normal gene, but also causing their G6PD to show a very low specific activity. In these individuals, the administration of strong oxidizing agents, such as members of the quinine-type class of anti-malarial drugs, can cause serious clinical complications, such as hemolytic anemia, since the reduced specific activity of their G6DP does not allow the production enough reducing agents to prevent unwanted rapid oxidative effects on your red blood cells. In areas where malaria infections are common and sometimes even epidemic, there is therefore a need for an effective rapid test that easily distinguishes people who have a reduced specific activity G6PD from people whose G6PD activity is normal, thus allowing the medical personnel ensure that (1) quinine anti-malarial drugs are only prescribed to individuals with specific or better G6PD activity and (2) people with less than normal G6PD activity are medicated with an alternative type of anti-G6PD activity malaria.

[048] In addition, several types of drugs are known to increase hemolytic anemia, in which the list includes, but is not limited to, anti-malarial drugs (primaquine, paquina, etc.), sulfonamides (sulfanilamide, sulfacetamide , sulfapyridine, sulfamethoxazole, etc.), sulfones (dapsone, etc.), nitrofurans (nitrofurantoin, etc.) and others (nalidixic acid, naphthalene, ciprofloxacin, methylene blue, toluidine blue, etc.).

[049] To test for G6PD deficiency, red blood cells that contain glucose-6-phosphate dehydrogenase (G6PD) are lysed on the test strip when they come into contact with the pre-treated sample application zone with blood lysis reagent. The G6PD in the lysed red blood cell reacts with its substrate on the pre-treated dry strip and the enzymatic reaction takes place to begin the color formation process in the presence of a tetrazolium dye and a hydride transfer agent. Catalyzed G6PD can oxidize glucose-6-phosphate to release NADPH and the released NADPH reduces the tetrazolium dye to change the color of the tetrazolium dye to formazan, such as purple or blue, etc., which can be detected visually on the same pad .

[050] According to a specific variant, the test strip is constructed in a single layer, where the sample application area and the color forming reaction area are on the same pad. The upper part of the test strip consists of an absorbent pad with absorbent material, if necessary. The test strip is pre-treated with lysis reagent, including ionic or non-ionic surfactants. A simple procedure for performing the test is provided, as there are no requirements for the blood lysis step before applying the sample. The pretreated test strip is impregnated with reagents, which comprise: glucose-6-phosphate which is a specific substrate for G6PD, coenzyme NADP or NAD, tetrazolium dye and hydride transfer agents, and a stabilizer, such as a sugar residue or a water-soluble polymer.

Rapid diagnostic test (RDT) and double test

[051] According to a variant, the present invention provides a rapid diagnostic test (RDT) in an anhydrous assay on an individual pad tape for point of care testing on site. Lateral chromatography in dry form provides a stable test device, a simple process and a quick test result. G6PD deficiency is a deficiency in the most common human enzymes affecting 400 million people and showing a high prevalence in endemic areas of malaria. This deficiency appears to provide some protection against infection with malaria. However, it can also cause hemolysis after administration of certain drugs against malaria, including PRIMAQUINE. As such, there is an urgent need for a rapid diagnostic test (RDT) suitable for the screening area.

[052] According to another variant, the present invention provides a test method in which the results of the G6PD RDT test using clinical samples can be correlated with the quantitative results of the test. The test can detect about <4 IU / gHb (normal 12 ± 2.09 U / gHb at 37 ° C), as deficient. The limit value can be changed depending on the tolerance of red blood cells against PRIMAQUINE in patients with G6PD deficiency. Accelerated stability studies indicated that the test strips were stable for three months at 45 ° C and for 10 days at 60 ° C. This RDT for G6PD can be combined with an RDT for malaria that is capable of detecting less than 30 parasites per pL of blood. This double test kit can diagnose malaria infection and G6PD deficiency at the same time using a small amount of blood (usually less than 10 pL). The test test is quick (<10 minutes) with a small amount of sample (<10 pL of blood), simple to use, economical and has no special storage requirements, which is a critical element for use on site.

Lateral flow chromatography

[053] Many of the “point of care” tests in place commonly used use a lateral flow assay based on membrane-based immuno chromatography, which benefits from the capillary action of microporous membranes. In lateral flow chromatographic tests, the substances to be analyzed in the specimen solution in the mobile phase are separated from the other components by binding affinity to capture molecules in solid stationary phases. The membranes, made of nitrocellulose or nylon, provide a matrix for the stationary solid phase of affinity chromatography and for the liquid phase of partition chromatography that leads to the immunocomplex particles that are to be separated from the other liquid solutes by capillary action .

[054] Microporous membranes, laid from nylon or nitrocellulose, have been used for antigen / antibody testing since about 1979, when it was first demonstrated that proteins could be transferred across a membrane. Nitrocellulose has been used in research techniques, such as Western blot and lateral flow immunodiagnosis. Microporosity and nitrocellulose offer many benefits for rapid immuno chromatography assays, including, for example, high binding capacity, non-covalent binding of proteins and a stable long-term immobilization environment.

[055] In general, in lateral chromatography, including assays conducted by enzyme, such as a test for G6PD deficiency, the anhydrous substrate is pre-deposited next to, or just behind, the junction of the sample receiving area on the chromatographic tape , i.e., individual pad tape, as in the present invention. However, it can be placed elsewhere in the sample flow path to accommodate particular requirements of the sample or one or more ingredients in the substrate, provided it is placed in the flow path long before the end of the “absorption pad”, where the sample flow is interrupted and any excess fluid present flows into the absorption pad or other drain device that may be provided.

[056] It is important that the dry substrate is deposited in an interval, preferably within a confined area, to facilitate that it is completely grasped by the sample flow. The placement of the dry substrate in the flow path should also take into account that the reconstitution of the substrate in a dissolved or dispersed form in the liquid sample must be complete by the time the sample reaches the point at which the sample flow ceases.

[057] For reasons of convenience of transport, storage and use, each chromatographic tape of the present invention can preferably be conditioned in a suitable device constructed so that the tape is positioned laterally. Many of these devices are well known in the art and any one that is constructed so that the performance of an assay on the chromatographic tape placed on it is performed by side flow can be used in an appropriate manner.

[058] According to a specific variant, the individual pad tape of the present invention provides the following characteristics, but without limiting this: 1. The individual pad tape comprises a porous matrix selected from nitrocellulose membrane, fiber glass, cellulose or polyester, etc. 2. The tape comprises an individual pad tape in which the sample application area and the deposition area for the dry reagent mixture of said tape are on the same pad, regardless of whether the two areas overlap or not. 3. The tape in the present invention has an individual liquid flow track for the reaction mixture that occurs after receiving the sample fluid. 4. The individual cushion tape of the present invention can optionally be pre-coated with a lysis reagent that contains an ionic (anionic, cationic and zwitterionic) or non-ionic surfactant and magnesium chloride, allowing the lysis of the biological fluid by contact the sample application area directly without pre-lysis with buffer. 5. The individual pad tape of the present invention contains a sugar residue selected from monosaccharide, disaccharide, polysaccharide or water-soluble polymer as a stabilizer to increase thermal stability. 6. The individual cushion tape of the present invention is formulated and differentially mounted on a device with a double test format to discriminate between moderate G6PD deficiency (for example: 10% -60% detection of normal activity) and severe G6PD deficiency ( for example: less than 10% detection of normal activity) when unarmed. The percentage (%) of activity for detection is not limited to 10% -60% or less than 10%. 7. The components in the reaction zone comprise enzyme substrates, reagents for color development and stabilizer. The individual pad system can be used for a quantitative or semi-quantitative test as the color change on the tape can be read by instruments, such as an optical spectrophotometer, digitizer, reader or densitometer. 8. The individual pad system is also applicable to an electrochemical system. The electron transfer from NADPH or NADH produced by G6PD to an electrode through oxireductase or an electron transfer mediator can be quantified using a dry electrode. 9. The individual pad system can be used for an immunoassay that immobilizes the captured molecules in solid phase.

Hemoglobin separation from the analyte by differential chromatography

[059] The key parameters that control signal strength in lateral chromatographic assays are capillary flow and the protein binding capacity of the membrane. Capillary flow and bonding capacity are determined by pore size, porosity and membrane thickness. The protein binding capacity of the membrane depends on its pore size and surface properties.

[060] The present invention provides chromatography in which hemoglobin is separated from the analyte using differential solid-phase chromatography. The color of the hemoglobin often interferes with the visual reading of the color of the reaction of the substance to be analyzed in the red blood cells. The present invention provides a method of lateral chromatography in which the different migration of the samples due to the difference in size, viscosity, precipitation of the analyte with reagent, difference in charge through the porous matrix impregnated with reagents that will interact with the analyte . The blood cells are applied to the sample zone, which is pre-treated with lysis agent for lysis of red blood cells in the reaction zone and the buffer is applied as needed. The lysed blood cells move to the reaction-results zone and mix with the reagent, the resulting composition remaining in the reaction-results zone, while the hemoglobin continues to travel, being separated from the resultant desired by such capillary strength. The separation of hemoglobin will make the detection of results through an indicator, such as color, more evident and easier to identify.

EXAMPLE

[061] A specific G6PD assay that can be easily used successfully by anyone working in a location, at home, in an office or in any location where trained laboratory personnel and instruments are not available, is specifically described below and the results are shown in figures 1 to 3 attached. Example 1. Preparation and testing of samples for G6PD deficiency test strips

[062] The pad was pre-treated with lysis buffer and dried at room temperature. The structures of the single and double-cushion tapes are shown in fig. 1. The pretreated pad was fixed on the plastic support and an absorbent paper was superimposed on the upper portion on the plastic support, like an absorbent pad. The test mixture containing glucose-6-phosphate, NADP, tetrazolium and a hydride or electron transfer agent was prepared and impregnated on the porous pad. The pad impregnated with test mixture was allowed to dry at room temperature overnight. • Preparation of the individual pillow: the pillow impregnated with the test mixture in the plastic support. • Preparation of the two pads: the pad for applying the sample is attached to the bottom of the test mixture - pad impregnated by overlapping 1-2 mm in the plastic support.

[063] A normal or deficient sample was applied to the tape. The result shows that normal samples certainly produce a stronger and more evident purple color in the individual pillow system of the present invention than in the two pillows, as shown in fig. 2. The deficient sample did not produce any color on both pads, the individual pad and both pads, as there was no G6PD available to produce NADPH. The result further shows that fluid migration is more uniform and faster in the individual pad than in the two pads and that the completion of blood cleaning is faster in the individual pad tape system of the present invention than in the two pads. Example 2. Preparation and testing of samples for lactate dehydrogenase test strips

[064] The pad was pre-treated with lysis buffer containing magnesium chloride and dried overnight at room temperature. The pretreated pad was fixed on a plastic support and absorbent paper was superimposed on the top of the pad on the plastic support as an absorbent pad. The test mixture containing lactate, NAD, tetrazolium and Tris-HCI pH 8.0 buffer, hydride transfer agent was prepared and impregnated on the porous pad. The pad impregnated with the test mixture was allowed to dry at room temperature overnight. • Preparation of the individual pillow: the pillow impregnated with the test mixture in the plastic support. • Preparation of the two pads: the sample pads are attached to the bottom of the test mixture - pad impregnated by overlapping 1-2 mm in the plastic support.

Exemplo 3. Preparação e realização de testes de amostras para fitas de teste de álcool Preparação das duas almofadas: as almofadas de amostra são ligadas à parte inferior da mistura de ensaio - almofada impregnada por sobreposição de 1-2 mm no suporte plástico.[065] Two microliters of blood sample were applied to this tape. The result shows that the LDH enzyme in the blood certainly produced a stronger purple color and more evident in the individual pillow tape system than in the two pillows, as shown in fig. 4. The result further shows that fluid migration is more uniform and faster in the individual pad than in the two pads and that the completion of blood cleaning is faster in the individual pad tape system of the present invention than in the two pads. pillows. The intensity of the purple color was measured using an aQzen reader as shown in Table 1. The intensity of the individual pillow is higher than that of the two pillow system, as shown in fig. 5.

Example 3. Preparation and testing of samples for alcohol test strips Preparation of the two pads: the sample pads are attached to the bottom of the test mixture - pad impregnated by overlapping 1-2 mm on the plastic support.

[066] The pad was pre-treated with buffer containing detergent and dried overnight at room temperature. The pretreated pad was fixed on a plastic support and absorbent paper was superimposed on the top of the pad on the plastic support like an absorbent pad. The test mixture containing alcohol, NAD, tetrazolium and Tris-HCl pH 8.0 buffer, hydride transfer agent was prepared and impregnated on the porous pad. The pad impregnated with test mixture was allowed to dry at room temperature overnight. • Preparation of the individual pillow: the pillow impregnated with the test mixture in the plastic support. • Preparation of the two pads: the sample pads are attached to the bottom of the test mixture - pad impregnated by overlapping 1-2 mm in the plastic support.

Exemplo 4. Preparação e realização de testes de amostras para fitas de teste de lactato desidrogenase - dependentes da atividade de lactato desidrogenase[067] Two microliters of blood sample containing alcohol was applied to this tape. The result shows that the presence of alcohol in the blood certainly produced a stronger and more evident purple color in the individual cushion tape system than in the two cushions, as shown in fig. 6. The intensity of the purple color was measured using an aQzen reader as shown in table 2. The intensity of the individual pillow is higher than that of the two pillow system, as shown in fig. 7.

Example 4. Preparation and testing of samples for lactate dehydrogenase test strips - dependent on lactate dehydrogenase activity

[068] The pad was pre-treated with buffer containing detergent and dried overnight at room temperature. The pretreated pad was fixed on a plastic support and absorbent paper was superimposed on the top of the pad on the plastic support like an absorbent pad. The test mixture containing alcohol, NAD, tetrazolium and Tris-HCI pH 8.0 buffer, hydride transfer agent was prepared and impregnated on the porous pad. The pad impregnated with test mixture was allowed to dry at room temperature overnight. • Preparation of the individual pillow: the pillow impregnated with the test mixture in the plastic support. • Preparation of the two pads: the sample pads are attached to the bottom of the test mixture - pad impregnated by overlapping 1-2 mm in the plastic support.

[069] Two microliters of sample containing lactate dehydrogenase (between 0.5 units and 6 units) were applied to this tape. The result shows that the activity of the LDH enzyme produced a stronger and more evident purple color in the individual pillow tape system than in the two pillows, as shown in fig. 8.

Claims (16)

1. Tape for a side flow chromatographic assay conducted by enzyme for a pre-selected analyte characterized by being made up of: an individual pad; and a substrate deposited as anhydrous form. wherein the individual pad is pre-treated with a lysis buffer; wherein the individual pad comprises a sample application zone and a resulting reagent zone, and the sample application zone and the resulting reactive zone are formed at the same location as the individual pad; wherein the substrate is capable of forming a precipitate when reacted with the pre-selected analyte; and wherein the pre-selected analyte is an enzyme; and wherein said strip is capable of separating said pre-selected analyte or reaction resulting from hemoglobin present in a blood sample under analysis by differential migration of said pre-selected analyte or components of hemoglobin; the red blood cells in said blood sample are lysed upon contact with said lysis buffer; said reaction result is a reaction product generated from the interaction between said pre-selected analyte and said anhydrous substrate; and said hemoglobin components migrate more quickly compared to said pre-selected analyte or said reaction result. 2. Tape, according to claim 1, characterized by the fact that the analyte is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase. 3. Tape, according to claim 1, characterized by the fact that the analyte is G6PD. 4. Tape according to claim 1, characterized in that the substrate is a test mixture comprising glucose-6-phosphate, an electron transfer agent or hydride and at least one between a nicotinamide-adenine phosphate -dinucleotide (NADP) or nicotinamide-adenine-dinucleotide (NAD). 5. Tape, according to claim 1, characterized by the fact that the substrate comprises an indicator. 6. Tape, according to claim 5, characterized by the fact that the indicator is a coloring compound. 7. Tape, according to claim 6, characterized by the fact that the coloring compound is selected from the group consisting of TTC (2,3,5-triphenyl-2H-tetrazolium chloride), INT (2- (4 -iodophenyl) -3- (4-nitrophenyl) -5- phenyl-2H-tetrazolium), MMT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) , XTT (2,3-bis- (2-methoxy-4-n-itro-5-su Ifofen i I) -2 H-tetrazolium-5-carboxanilide), NBT (Nitroblue tetrazolium), MTS (3- ( 4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) and DCPIP (2,6-dichlorophenolindophenol). 8. Tape according to claim 6, characterized in that the coloring compound is a tetrazolium salt. 9. Side flow chromatographic assay conducted by enzyme for a pre-selected analyte, characterized by understanding; (a) providing a test device comprising a tape, as defined in claim 1; (b) applying a sample to the individual pad on which the substrate is deposited; (c) allowing the sample to flow through the individual pad to reach the substrate; (d) allowing the sample to react with the substrate to form a precipitate; and (e) identify and interpret the response to indicate the presence or concentration of the analyte in the sample. 10. Assay, according to claim 9, characterized by the fact that the analyte is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase. 11. Assay according to claim 9, characterized in that the substrate is a test mixture comprising glucose-6-phosphate, a hydride or electron transfer agent and at least one of nicotinamide-adenine-dinucleotide (NADP ) or nicotinamide-adenine-dinucleotide (NAD). 12. Assay according to claim 9, characterized in that the substrate comprises an indicator. 13. Assay, according to claim 12, characterized by the fact that the indicator is a coloring compound. 14. Assay according to claim 13, characterized in that the dye compound is selected from the group consisting of TTC (2,3,5-triphenyl-2H-tetrazolium chloride), INT (2- (4- iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium), MMT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide), XTT (2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide), NBT (Nitroblue tetrazolium), MTS (3- (4,5-dimethylthiazol-2-yl ) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) and DCPIP (2,6-dichlorophenolindophenol). 15. Assay according to claim 13, characterized in that the coloring compound is a tetrazolium salt. 16. Chromatographic assay of lateral flow conducted by enzyme to detect the presence of G6PD, characterized by the fact of understanding; (a) providing a test device comprising an individual pad tape, as defined in claim 1: (i) an individual pad pretreated with lysis buffer; (ii) a test mixture comprising glucose-6-phosphate, a hydride transfer agent and at least one of nicotinamide-adenine-dinucleotide (NADP) or nicotinamide-adenine-dinucleotide (NAD) phosphate, and (iii) a tetrazolium salt, (b) applying the sample to the individual pad; (c) allowing the sample to flow through the individual pad to contact the test mixture; (d) allowing the sample to react with the test mixture to produce a precipitate; and (e) identify and interpret the response to indicate the presence or concentration of the analyte in the sample.

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